Project Summary/Abstract Higher organisms have evolved sophisticated mechanisms to regulate gene transcription by RNA polymerse II (Pol II) in response to a variety of developmental, environmental, and nutritional cues. Improper transcription regulation can lead to developmental defects and disease states. Our past studies of Drosophila melanogaster heat shock (HS) genes revealed that promoter-proximal pausing during early elongation was a rate-limiting and regulated step in transcription, but this stood in contrast to the canonical model of regulation at recruitment of the preinitiation complex (PIC). However, recent genome-wide studies in metazoans revealed a large fraction of gene regulation occurs after PIC formation during early elongation providing a paradigm shift. In this proposal, I focus our efforts on understanding the mechanisms underlying promoter-proximal pausing and regulated escape into productive elongation. Our overall approach is to examine Pol II pausing and regulated escape at very high resolution in vivo under normal conditions and after specifically disrupting promoters, transcription factors (TFs) or TF interactions. In Aim 1, we employ a novel nuclear run-on method, called Precision GRO-seq (PRO-seq), to obtain high spatial (base pair) and temporal (minute) resolution data that quantitatively tracks Pol II pausing and its regulated escape into productive elongation in Drosophila during a time course of transcription activation. These genome-wide data will be used to refine existing, and generate new, hypotheses for the mechanisms that contribute to regulation. In Aim 2, the roles of DNA sequences, specific TFs, and nucleosomes in regulation will be tested by applying disruption or inhibition strategies and evaluating changes in transcription and TF interactions at gene promoters in vivo using PRO-seq and other high resolution biochemical methods (e.g., ChIP-exo) and optical sectioning of live polytene nuclei. In addition to standard mutant and RNAi strategies, we will use well-defined drugs to specifically inhibit steps in initiation or pause escape, and also make use of a novel inhibitory RNA aptamer technology, expressing high affinity aptamers against key TFs to bind and inhibit their macromolecular interactions. In Aim 3, we will again use PRO-seq and other genome-wide, high-resolution, methods to examine the effects of targeted perturbations by directing TALE- hybrids (engineered DNA binding domains fused to TFs or TF domains) to endogenous genes. Finally, because Aims 1-3 focus on the efficient and economical Drosophila systems, Aim 4 tests key hypotheses, which were developed and tested in Drosophila, in human tier1 ENCODE lines and mouse embryonic stem cells.